Fully automated viability and toxicity screening—A reliable all‐in‐one attempt

Abstract Background The CRISPR/Cas9 technology is nowadays a common tool for genome editing to achieve new insights into, for example, diagnostics and therapeutics in cancer and genetic disorders. Cell proliferation and anticancer drug response studies are widely used to evaluate the impact of editing. However, these assays are often time‐consuming, expensive, and reproducibility is an issue. To overcome this, we developed a fast and cheap assay that combines a fully automated multispectral fluorescence microscopy platform with a nuclei staining and open‐source software analysis. Methods Here, we generated different LEDGF/p75 model cell lines to validate the effect on proliferation and chemosensitivity. Therefore, a fast protocol for an optimized all‐in‐one attempt for cytotoxicity screenings and proliferation analysis of adherent cells in a 96‐well plate format was established using differential staining with two fluorescent dyes (Hoechst 33342 and propidium iodide) for live/dead cell discrimination. Subsequently, an automated cell nuclei count and analysis were performed using bioimage informatics. Results With the new established assay technology, up to 50,000 cells/well can be detected and analyzed in a 96‐well plate, resulting in a fast and accurate verification of viability and proliferation with consistency of 98% compared to manual counting. Our screening revealed that LEDGF depletion using CRISPR/Cas9 showed a diminished proliferation and chemosensitivity independent of cell line origin. Moreover, LEDGF depletion caused a significant increase in 𝛾H2AX foci, indicating a substantial increase in DNA double strand breaks. LEDGF/p75 overexpression enhanced proliferation and chemoresistance underlining the role of LEDGF in DNA damage response. Conclusion Independent of cancer cell type, LEDGF/p75 is a central player in DNA damage repair and is implicated in chemoresistance. Moreover, our automated fluorescence biosensor technology allowed fast and reliable data acquisition without any fixation or additional washing steps. Additionally, data analysis was implemented using the modular open‐source software that can be adapted as needed.


| INTRODUCTION
Genomic alterations can cause cancer, and with the genome editing tool CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated enzyme 9), a technology has been developed to explore genetic changes in a fast and easy way. 1,2The identification of potential drivers for cancer, as well as potential therapy targets, has been already accelerated through the introduction of RNA interference (RNAi) but CRISPR/Cas9 technology is speeding up the research advances even further.In comparison to other genome editing tools such as transcription activator-like effector nuclease (TALEN) or zinc finger nucleases, CRISPR/ Cas9 is efficient, easy programmable, modifies chromosomal targets with high fidelity.It offers the possibility of multiplex genome editing as well as transcriptional regulation. 3Moreover, CRISPR/Cas9 technology allows not only the knockout (KO) of genes and gene modulation but also directed knockin of gene expression cassettes into a safe harbor locus. 4 major obstacle in cancer treatment is chemoresistance.The list of involved genes is long and can also vary depending on the type of cancer.The transcriptional co-activator lens epithelium derived growth factor, LEDGF (also known as dense fine speckled autoantigen of 70 kDa, DFS70), is a multi-functional chromatinbinding protein and its longer splice variant LEDGF/ p75 is known to be overexpressed in different types of cancer, for example, breast and prostate cancer cells. 5,68][9] During stress conditions, LEDGF/p75 binds to stress-response elements (STREs) to activate the expression of stress-related genes, resulting in increased chemoresistance. 6 robust drug sensitivity assay is a prerequisite to establish a reliable drug efficacy and potency.Typically, proliferation and cytotoxicity for example, sensitivity towards chemotherapeutics are assessed by colorimetric assays measuring the cell content or metabolic activity.Sulforhodamine B (SRB) stains the cellular protein content and is, since 1990, a widely used and cost-efficient assay. 10,11The binding of the dye is stoichiometric, and its intensity is proportional to the cell mass.Furthermore, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra zolium bromide) assay, which measures mitochondrial NADH dehydrogenase activity, and the ATP assay (which uses, e.g., luciferin to evaluate ATP level), are frequently used metabolic assays.Nevertheless, the reproducibility is often challenging due to many fixing, staining, and washing steps that might cause cell detachment or incomplete excess dye removal.Moreover, most of these assays are time-consuming.The aforementioned problems that arise by the usage of colorimetric assays can be overcome by the deployment of automatic analysis of image data.In addition, manual assessment of data is prone to error due to the subjectivity of the assessor.
Here we propose a strategy that combines fluorescence staining with bioimage informatics to access cell viability and cytotoxicity in an automated, high-throughput manner.Using bioimage informatics, extensive image datasets can be analyzed fast and reproducibly. 8,12][19] Additionally, CRISPR/Cas9 technology can be used to modulate the expression of oncogenes and its impact on the chemosensitivity.In a previous study, we established already LEDGF-modified HEp-2 cell lines which showed an increased chemoresistance with LEDGF/p75 overexpression (LEDGF/p75 OE) and a reversed effect in LEDGF KO cells. 8To provide further evidence regarding the changes in chemosensitivity we designed an assay setup to analyze the effect of LEDGF KO and LEDGF/p75 OE in different cell lines.

| Antibodies
The following antibodies were used in this study: anti- H2AX (Cell Signaling Technology Cat# 2577, RRID: AB_2118010, Massachusetts, USA), anti-C-LEDGF/ p75 (Bethyl-Laboratories, Cat# A300-848A, RRID:AB_2171223, acquisition without any fixation or additional washing steps.Additionally, data analysis was implemented using the modular open-source software that can be adapted as needed.

| Generation of LEDGF-modified cell clones
CRISPR/Cas9-modified HEp-2, U2OS, and LoVo cells were generated according to the method described by Liedtke et al. 8 Briefly, cells were seeded in six-well plates (Th.Geyer, Renningen, Germany), incubated for 24 h and subsequently transfected with px458_sgR_DFS70_E1 using Lipofectamine™ 3000 according to manufacturer's instructions (Thermo Fisher Scientific, Massachusetts, USA).For the EGFP-LEDGF/p75 expression, LEDGF KO cells were co-transfected with px458_sgRNA_AAVS1 and pAAVS1_CAG-EGFP-LEDGF/p75. Transfected cells were enriched by EGFP selection via FACS using S3e cell sorter (Bio-Rad).Fluorescent, single cell colonies were picked after 7-10 days to establish LEDGF/p75 re-expressing cell lines.Obtained cell clones were analyzed to verify the integration of the expression cassette at the AAVS1 locus by PCR as described in Oceguera-Yanez et al. 4 showing only heterozygous integrations.The expression level of LEDGF/p75 was verified by western blot analysis due to relatively quantification of protein bands by ImageJ. 21

| Proliferation analysis
Cells were seeded at 5 × 10 3 cells/well into multiple 96well plates (Th.Geyer, Renningen, Germany) and incubated for 24-96 h at 37°C and 5% CO 2 .To determine the accuracy of cell seeding, one plate was used as a 0 h control.For proliferation analysis after 0-96 h, the corresponding plates were stained as explained in Section 2.7 and analyzed as explained in Section 2.8.
2.8 | Digital image analysis and bioimage informatics Analysis of proliferation and cytotoxicity assays after Hoechst/PI staining was performed using the fully automated multispectral fluorescence microscopy VideoScan platform 13,22 In principle, and for users who do not possess the VideoScan system, our all-in-one attempt is compatible with common fluorescence microscopes.PI/Hoechststained cells were measured using an exposure time of 0.5-1 s.Each well of the 96-well plate was dissected into 12 subunits, imaged in meandering sequence at 10× magnification and reassembled as a single image.Images were subsequently analyzed using bioimage informatics as follows.All image processing steps were performed using the library scikit-image v.0.17.2.The obtained images were converted to grayscale and all parts of the image outside the analyzed well were masked using the image average to not interfere with detection.Additionally, the image was slightly smoothed before detection using median filtering (4 × 4 neighborhood; skimage.filters.median)to reduce the noise.Cell count per image was determined via Laplacian of Gaussian Blob detection as implemented in scikit-image skimage.feature.blob_log;v.0.17.2; following settings: min_sigma: 1.5, max_sigma: 4, num_sigma: 10, threshold: 0.005, overlap: 0.35, exclude_borders: False.To exclude dead cells, the PI-positive cells were subtracted from the number of Hoechst-positive cells.Prominent problems during image acquisition and data analysis were addressed in a table in (Supplement 1 and 2-Data S1).

| Sulforhodamine B assay
To determine cell proliferation, cells were seeded at a density of 5 × 10 3 cells/well in a 96-well plate (Th.Geyer, Renningen, Germany) and a SRB assay was performed according to nature protocols. 11Briefly, 96-well plates were fixed with 10% ice cold TCA solution for 1 h at 4°C, followed by washing 4× with slow-running tap water.Plates were allowed to air dry and 0.057% SRB solution was added directly into the wells and incubated a RT for 30 min.Plates were rinsed again 4× with tap water containing 1% acetic acid and allowed to air dry at room temperature.Tris-base solution (10 mM, pH 10.5) was added directly into the wells and incubated on a shaker for 5 min to solubilize the protein-bound SRB-dye.Measuring was performed by OD analysis at 510 nm.

| Statistical analysis
All data were statistically analyzed with the statistical computing language R ≥ 4.2.1. 24The Kolmogorov-Smirnov test was used for testing normal distribution.To control the α error inflation, the Bonferroni correction is applied or Tukey's HSD test (stats::TukeyHSD()) is used to test the differences between the mean values of the sample for significance.Thereby, the stats::TukeyHSD() function uses the Tukey-Kramer procedure 25 p < 0.05-were considered as significant.Experiments were conducted with at least three biological replicates.Data were further analyzed using RKWard v. 0.7.5 26 for the R statistical computing environment using the packages visreg v. 2.7.0 27 and report v. 0.3.5.A linear model was estimated using ordinary least squares (OLS).The foci count data were processed using the countfitteR 28 package to determine the distributions and mean values.Data were further analyzed using RKWard v. 0.7.1z + 0.7.2 + devel2 13 for the R statistical computing environment.Dose-response curves were fitted (95% confidence interval) with multiparametric functions (EXD3: Three-parameter exponential decay model; LL4: Four-parameter log-logistic model) from the drc package. 29The optimal model was selected by using the Akaike information criterion (AIC) as criterion.

| Software enables reliable quantitative cell counting with life-death discrimination for cytotoxicity screenings
So far, we have used the SRB assay for proliferation and chemosensitivity analysis, but this assay is timeconsuming and requires several washing steps.Moreover, a fixation step is needed, which can cause dislodging of adherent cells.To achieve a good reproducibility, a higher number of replicates was often necessary.Therefore, a fast fluorescence-based approach using Hoechst/PI staining of non-fixed, living cells in combination with an automated microscopy platform was established (Figure 1).
The Hoechst/PI staining allows a cell count (rather than protein content or metabolic activity) as well as live/dead discrimination.As representative shown in Figure 2 for HEp-2 cells, cells should be evenly distributed (Figure 2A), and stained properly with Hoechst and PI to allow single cell detection (Figure 2A,B) even after 96 h.We applied a Python script 30 for automatic cell counting.The enlarged image sections show the precision of the software, which was tested on U2OS, HEp-2, and LoVo cells.Detection limits can be considered below 500 and above 50,000 cells per 96 well, depending on cell morphology, due to insufficient focus points or clumpy cell formation.
To verify the accuracy of automated detection, approximately 25,000 DAPI-stained cells were also manually counted.To ensure accuracy during manual counting, the image of the well was divided into subareas (Figure 2C).Representative subareas were analyzed and counts are shown in Figure 2D.Thereby, both counts (manually counted: 25,125 cells, detected by software: 25,693 cells) differ 2% from each other (Figure 2) and can be considered as comparable methods.We fitted a linear model (estimated using OLS) to predict manual counting with software counting (formula: manual ~ software).The model explains a statistically significant and substantial proportion of variance (R 2 = 0.99, F (

| Automated image analysis reveals: LEDGF/p75 expression affects proliferation and chemoresistance in a range of cell lines
After accuracy verification of the software, we examined genetically LEDGF-modified U2OS, HEp-2, and LoVo cell lines regarding their proliferation capacity and chemosensitivity.Here, initially, we compared the SRB and PI/Hoechst staining assays with each other.In comparison to the automated image analysis software, SRB assays showed no significant difference in growth behavior (Figure 3), if only three technical replicates are used.
In the VideoScan analysis, the investigated WT cell lines exhibited different proliferative capacities while U2OS showed only a threefold increase in cell number after 72 h, LoVo and HEp-2 cells showed a fivefold and sevenfold increase, respectively (Figure 3A).LEDGF KO significantly reduced the proliferation rate in faster proliferating LoVo and HEp-2 cells, while in U2OS cells only after longer cultivation (96 h) the proliferation in LEDGF KO cells stagnated in comparison to the WT.As expected, the proliferation analysis showed that LEDGF/p75 overexpression cells are proliferating significantly faster than LEDGF KO cells, correlating with longer cultivation time independent of the cell type; however, this effect was less pronounced in slower dividing U2OS cells (Figure 3).LEDGF OE in HEp-2 cells was strong also in comparison to the WT expression level (Figure 6D) which was reflected by a significantly enhanced proliferation in comparison to the WT (Figure 3B).In addition, the chemosensitivity towards etoposide regarding the LEDGF status was investigated (Figure 4).As there was no difference in growth behavior between untreated cells and cells treated with DMSO, untreated cells were used as control for calculation.A low etoposide concentration (5 μM) already resulted in 40%-60% killing rate after 48 h in all WT cells.Similar to the proliferative behavior, LEDGF KO caused a significant increase in the killing rate as compared to the WT cells; however in U2OS, cells this effect was diminished and only after 72 h etoposide visible.This enhanced chemosensitivity was effectively reversed by overexpressing LEDGF/p75.Moreover, LEDGF OE resulted partially, even, in a significantly increased chemoresistance.As already shown in the proliferation study, the SRB assay was able to detect increasing killing rates in all cell lines (Figure 4D-F), but no significant difference dependent on the LEDGF expression level.

| Knockout of LEDGF/p75 renders cells more chemosensitive towards etoposide
The median effective dose (ED 50 ) was determined to draw conclusions about the efficacy of etoposide as a chemotherapeutic agent in LEDGF KO or LEDGF/ p75 knockin mutations in various reference and colorectal cancer cell lines.While FACS analysis showed much higher overall ED 50 values in HEp-2 cells and no difference between HEp-2 WT and HEp-2 LEDGF/ p75 OE cells, automated image analysis software acted more sensitive and detected lower overall ED 50 valued and a significant difference in resistance when comparing HEp-2 LEDGF/p75 OE cells with HEp-2 WT cells (Figure 5A).The analysis of the LoVo cell lines showed F I G U R E 1 Workflow for automated fluorescent-based proliferation and cytotoxicity screening.(A) After counting, cells are seeded at appropriate cell densities into 96-well plates.(B) Compound of interest is added directly to the cells without washing.Include compound solvent and medium only control.Check possible autofluorescence of the compounds ahead of the experiment!(C) After treatment, Hoechst/PI-solution is directly added to the cells in the well.If background fluorescence is detectable, a washing step with 1x PBS is required.(D) Pictures of each well are taken by a fully automated imaging platform (e.g., VideoScan), followed by digital image analysis (by e.g., VideoScan analyzer).Determination of growth and, if applicable, ED 50 is determined by data analysis and statistical analysis.that the ED 50 values for all three cell lines are very close to each other and that significant but weak differences can be observed, whereas the analysis using automated image analysis shows a well separated and significantly different ED 50 distribution between the WT, the LEDGF KO and the LEDGF/p75 overexpressing cell lines (Figure 5B).Looking at the analysis of the ED 50 values for the U2OS cells, it is noticeable that the FACS analysis does not show any significant differences between U2OS WT and U2OS LEDGF/p75.The automated image analysis software can clearly show this small but significant difference (Figure 5C).

Cas9-modified LEDGF cancer cell models
The generation of CRISPR/Cas9-modified LEDGF Hep-2 cell models has been described previously. 8Additionally, LEDGF-modified U2OS and LoVo cell lines were generated.To achieve a complete LEDGF KO, a specific sgRNA targeting exon 1 of PSIP1 gene was designed (Figure 6A).Thereby, the transient transfection with a non-viral px458_DFS70_E1 vector allowed an enrichment of Cas9positive cells via FACS sorting using EGFP as a marker.Subsequently, LEDGF/p75 expression cassette was integrated at the human safe harbor locus AAVS1 (directed integration to avoid nonspecific side effects) using CRISPR/ Cas9 (Figure 6B).Cell lines were established from singlecell clones and complete LEDGF KO was verified on protein level (Figure 6C,D) and on genomic level (Supplement 4-Data S1).The robust EGFP-tagged LEDGF/p75 expression and correct subcellular localization (in the nucleus) was determined by immunofluorescence (Figure 6C) and immunoblotting (Figure 6D), where LEDGF/p75 OE cells show an up to 10-fold increased LEDGF/p75 expression, compared to the expression in WT cells (Figure 6E).Note, the membrane was stripped between LEDGF/p75 and αactinin protein detection and to allow an appropriate identification of EGFP-LEDGF/p75 expression, an antirabbit secondary antibody was used, whereas an antimouse antibody was used for the detection of αactin expression.In LoVo cell lines, an anti-βtubulin antibody was used due to low detection level 6D).
| LEDGF KO causes an increase in

DNA damage
LEDGF is involved in DNA damage repair, and we have previously shown that upon LEDGF KO, the H2AX foci formation is markedly increased, even without chemotherapeutic treatment. 8H2AX is the marker for DNA double-strand breaks (DSB), the most severe DNA lesion.Therefore, we determined the H2AX foci formation in the investigated cell models.The immunofluorescence analysis (Figure 7A-C) revealed significant upregulated expression of H2AX in all LEDGF KO cells compared to the corresponding WT cells in HEp-2 (3-fold, p WT-KO = 0.005), U2OS (2.6-fold, p WT-KO = 0.002) and LoVo (10-fold, p WT-KO = 8.5 × 10 −6 ) cells.This effect can be significantly reversed by LEDGF/p75 overexpression (Figure 7D-F).

| DISCUSSION
Proliferation analysis and cytotoxicity screenings are standard procedures in medical research.The demand on the tests is that they are reliable, fast, and easy to handle.Plenty of methods are available, but many of them have drawbacks.The routine use of colorimetric staining assays like NRU assay, 32 crystal violet assay, 33,34 and SRB assay 11 (Supplement 5-Data S1) often leads to inconclusive results with high standard deviations, caused by excess binding of dyes, incomplete washing steps and a low sensitivity.In addition, these assays stain any proteins that are present.Alternatively, metabolic activity assays, such as the MTT/ MTS, XTT, LDH, WST assays [35][36][37][38][39] or fluorometric assays like the almarBlue assay, 40,41 the CFDA-AM assay, 40,42 and the Annexin V assay 43 can be used (Supplement 5-Data S1).These are also colorimetric assays, but detection depends on the conversion of added chemicals which correlates with the amount of mitochondria in a given cell and activity of cellular metabolism.Therefore, these assays cannot be used for comparison between different cell lines.Nevertheless, metabolic assays show an increased cytotoxicity, chemicals may interfere with possible tested drugs and again no quantitative statement can be made about the corresponding cell number.Another accurate possibility for analysis of cytotoxicity can be the analysis of exposed phosphatidylserine residues on the cellular membrane using the Annexin-V assay.
During apoptosis, phosphatidylserine flips from the inner cell membrane to the outer cell membrane, and the amount corresponds to early or late apoptosis.Due to fluorescence labeling, these events can be tracked by a flow cytometer or fluorescence microscope, allowing the detailed analysis of each single cells 44 (Supplement 5-Data S1).Nevertheless, all protocols contain many individual washing and staining steps which lead to multiplying errors.Moreover, many assays are not only time-consuming but also cost intensive.
To tackle this problem, we propose a fast and cheap protocol for a straightforward all-in-one staining and detection procedure (protocol in Supplement 1-Data S1).Our assay requires no washing step, is independent of metabolic activity or amount of mitochondria, no additional fluorophore structures need to be added.There is no need to worry about interference with chemical substances, Hoechst/PI solution is directly added to living adherent cells with a high sensitivity of at least 500 cells/well of a 96-well plate and subsequently stained nuclei are detected by automated microscopy and images are processed by software packages for viable cell quantification.In principle, and for users who do not possess the VideoScan system, our all-in-one attempt is compatible with all common fluorescence microscopes with and without automatic stage.After the detection procedure cells can even be used for further analysis like indirect immunofluorescence analysis.To verify the accuracy of our software, we performed a comparison between our program and manually counted cells, which resulted in a consistency of almost 98% of counted single cells (Figure 2).Regarding F I G U R E 5 ED 50 determination of different cell lines using FACS analysis and automated image analysis software.Determination of ED 50 value was performed on wildtype (WT), LEDGF knockout (KO), and LEDGF/p75 (OE) cells, treated with indicated concentrations of etoposide for 48 h etoposide, followed by 3 days of recovery.Viable (A) HEp-2 cells, (B) LoVo-cells, and (C) U2OS cells were analyzed by the digital image analysis software (upper histograms) whereas a parallel run was analyzed by FACS (lower histogram).Dose-response curves were fitted with multiparametric functions from the drc package. 29,31The optimal model was selected by using the AIC as criterion.The triangles on the x-axis represent the dose of etoposide needed to reach ED 50 .*p < 0.05.
the commercially available CCK-8 assay, which is one of the most used assays for cell proliferation and cytotoxicity, [45][46][47] our assay is 42 times less expensive (CCK-8 kit: 0.67 €/1 reaction, our assay: 0.016 €/1 reaction), more sensitive-we only need half of the cells to exceed the detection limit and not limited by metabolic activity of the cell.Our approach can make semi-quantitative statements, such as colorimetric assays, but can also accurately quantify the total number of cells and the number of dead cells simultaneously.Other protocols use fixed cells 48 for the analysis of DNA bound dyes while we use living cells where Hoechst stains all nuclei, but PI incorporates only in late apoptotic or necrotic cells whose cell membrane is permeable.This procedure can serve for a wide range of experiments like multiplex testing of different drugs to calculate ED 50 level of different compounds, analyzing the increase/decrease in cytotoxicity after genomic modulation of cells (e.g., after knock-out or knock in of certain genes).It can be also used for checking proliferation of cells prior to experiments or comparing proliferation of different cell lines with potential new anticancer drugs.
In our study, the newly established assay was used to pursue the investigation of CRISPR/Cas9-generated LEDGF KO and LEDGF/p75 overexpressing cell lines and their chemosensitivity.LEDGF/p75 has been reported to be overexpressed in different tumor and cancer cell lines 7,49 and to promote repair of DSBs by the homologous recombination repair pathway. 9In our previous studies, we underlined the crucial role of LEDGF as a main player in initiating DNA repair processes. 8The lack of LEDGF expression resulted in an accumulation of H2AX, 8 an early responder of the DNA repair and marker for DSBs. 50In the present study, H2AX was significantly increased (depending on cell line between 2.6-and 10.5-fold, Figure 6) in LEDGF KO cells independent of the cell line, whereas LEDGF/p75 OE was able to rescue the WT phenotype with only background H2AX foci.Interestingly, H2AX is also an indicator for genome integrity and persistent H2AX foci formation is also related to tumorigenesis and genome instability. 51,52This may suggest that LEDGF participates in the maintenance of genome integrity.Moreover, depletion of SETD2, the only methyltransferase catalyzes the tri-methylation of histone-h3 lysine-36 (H3K36me3), also causes persistent H2AX foci. 52,53The histone reader LEDGF preferentially binds to H3K36me3 and its chromatin binding is reduced upon SETD2 depletion. 9Additionally, our group found out, that persistent H2AX foci relate to insufficient degradation of H2AX due to interaction of LEDGF/p75 with the nuclear proteasome activator PA28. 8The increased H2AX foci correlated with decreased proliferation rates in all LEDGF KO cells.This effect was reversed upon LEDGF overexpression and for strong LEDGF OE, the proliferation rate was even more elevated than in the WT cells.In breast cancer, upregulation of LEDGF/p75 expression also resulted in increased proliferation rates. 54dditionally, chemosensitivity towards etoposide depending on the LEDGF status was investigated.As expected, LEDGF KO induced a sensitizing effect, which indicates that increased H2AX is related to genomic instability and the cells are less stress resistant.On the contrary, LEDGF/p75 OE not only reversed this sensitizing effect, but partially led to an even higher chemoresistance against etoposide.Previously, chemoresistance has been described to be correlated with increased LEDGF/p75 expression in prostate cancer. 6Furthermore, the higher chemoresistance of LEDGF/p75 OE might also be related to the role of the shorter splice variant LEDGF/p52, which plays rather a pro-apoptotic role and is considered as a counterpart of antiapoptotic LEDGF/p75. 55In fact, WT cell lines most likely express both splice variants LEDGF/p75 and LEDGF/p52.However, we only verified the expression of the longer splice variant p75 (Figure 5D).Nonetheless, LEDGF sgRNA targets exon 1 and thereby knocks out both splice variants.LEDGF/p75 OE model was established using LEDGF KO cells and therefore potential LEDGF/p52 expression was not reconstituted.Therefore, we cannot exclude that LEDGF/ p52 might counteract LEDGF/p75 in WT cells leading to an altered proliferation and chemosensitivity.Nonetheless, specific depletion of LEDGF/p52 had no effect on chemosensitivity towards camptothecin. 9 F I G U R E 6 Generation of CRISPR/Cas9-modified LEDGF cancer cell models.(A) Scheme of LEDGF knockout generation: Specific sgRNA for Exon 1 of LEDGF-coding gene (PSIP1) was designed to introduce a target-specific DNA double-strand break (DNA DSB) with CRISPR/Cas9 technology.Due to endogenous error-prone non-homologous end joining (NHEJ) pathway, indel mutations can cause a frameshift leading to premature stop codons and subsequent gene knockout (LEDGF KO).(B) Scheme of LEDGF/p75 knockin generation: LEDGF overexpressing cells were created by introducing a DNA DSB at genomic safe harbor locus AAVS1 using an AAVS1-specific sgRNA and an HDR template containing EGFP-LEDGF expression cassette (ELC) flanked by homologous arms (HA) to the cutting site at the AAVS1 locus.After the induction of a DSB, homology-directed repair (HR) pathway mediates the integration of the ELC. and editing (equal).Lysann Schenk: Conceptualization (equal); funding acquisition (equal); methodology (equal); resources (equal); supervision (equal); validation (equal); writing -review and editing (equal).

2
Detection of stained nuclei by digital image analysis software and comparison of cells counted manually and by digital image analysis software.(A) HEp-2 cells stained with Hoechst and counterstained with PI directly after seeding (0 h) and (B) after 96 h of growth.Images were taken by a fully automated microscopy platform, nuclei are visible as light gray spots and can be overlayed.Digital image analysis software uses Laplacian-of-Gaussian Blob detection, as implemented by the skimage library.Each detected nucleus is marked with a red circle.To determine cell viability, cells stained with PI are subtracted from the total number of Hoechst-positive cells.Scale bar = 100 μm.(C) Segmentation of an image taken from one well of a 96-well plate.Inner subareas (I and II) were used for comparison between manual and automated analysis.(D) An amount of 20 inner subareas (indicated by colored dots) of a 96-well plate cultured with Hoechst-stained HEp-2 cells were analyzed in 10× magnification.The amount of manually (manual) counted HEp-2 cells was plotted versus the number of cells counted by digital image analysis software (software).